PROJECT SUMMARY/ABSTRACT Seasonal influenza is associated with up to 650,000 respiratory deaths per year worldwide. Influenza A virus injures the lung to cause the acute respiratory distress syndrome (ARDS). Influenza A virus-induced ARDS carries a mortality rate approaching 40% despite advances in anti-viral therapies and supportive care, with most patients succumbing to intensive care unit (ICU) complications as they recover from the initial infection. In mice, dysregulated repair leads to widespread and persistent alveolar epithelial abnormalities following severe influenza A. We reason that activation of repair pathways during recovery from severe influenza will shorten the duration of time that patients require the ICU, thus mitigating the ICU?s attendant morbidity and mortality. CD4+Foxp3+ regulatory T (Treg) cells are required to coordinate resolution of lung inflammation and repair of lung damage in mouse models. These cells appear in the alveolar spaces of patients with ARDS and display epigenetic and transcriptional profiles predicted by murine experiments. In the injured lung, Treg cells exert myriad pro-recovery functions, including generation of pro-epithelial molecules such as amphiregulin, the loss of which worsens influenza A-induced acute lung injury in mice. Within Treg cells, the DNA methylation pattern at specific genomic loci controls their identity and suppressive function. The epigenetic regulator protein Uhrf1 plays an essential role in maintaining cell type-specific DNA methylation signatures. The necessity of Uhrf1 in maintaining Treg cell identity and pro-repair function during the recovery phase of influenza A remains unknown. Likewise, the necessity of amphiregulin in inducing healthy epithelial repair during the recovery phase is also undefined. We hypothesize that Treg cells require Uhrf1 to maintain their pro-repair function and amphiregulin to induce epithelial repair during recovery from severe influenza A virus infection. We propose three Specific Aims, which use innovative approaches to test our hypothesis, including cutting-edge murine systems, novel computational platforms, and a human case-control study that will translate our findings to the bedside. Aim 1 will determine whether Uhrf1 is necessary to maintain Treg cell transcriptional programs and pro-repair function during recovery from influenza A. Aim 2 will ascertain the necessity of Treg cell-generated amphiregulin in promoting repair during recovery from influenza A-induced lung injury. Aim 3 will determine whether transcriptional and epigenetic signatures in alveolar Treg cells are associated with 30-day mortality in selected patients with severe viral pneumonia. Our proposal will establish causal evidence linking drug- targetable mechanisms to detailed physiologic readouts. Elucidating these causal links will inform the development of pro-recovery therapeutic approaches for severe influenza and other causes of ARDS.